Perfringolysin 0 (PFO), a cytolysin (Mr 54,000) produced and secreted by Clostridium perfringens, belongs to a family of related cytolysins now termed the cholesterol-dependent cytolysins (CDCs) and is produced by a variety of Gram positive pathogenic bacterial species. PFO typifies the CDCs, with a hydrophilic primary structure that ultimately forms a cytolytic membrane complex. After binding to the target membrane, PFO monomers oligomerize into supramolecular complexes and lyse the cell. During the current grant period, we identified the regions of PFO that form the aqueous-membrane interface and determined that each monomer inserted two B-hairpins into the bilayer to form the B-barrel of the pore. We also found that PFO forms a prepore complex prior to the insertion of these domains. The studies herein are designed to further our understanding of the mechanism by which these intriguing toxins alter their structure and interact with one another and the membrane surface as they make the transition from a soluble monomer to a membrane-bound oligomeric complex. The specific aims of this proposal are to: 1) Determine the topography of PFO relative to the membrane and identify intramolecular conformational chances at different sta2es of pore formation. 2) Elucidate the interactions between transmembrane B-hairpins in the oligomer. 3) Identify the PFO residues involved in subunit-subunit interactions. 4)Identify the nature of the intermedilysin receptor. Aim I will be accomplished by the use of fluorescence resonance energy transfer (FRET) to measure distances from various points in the PFO structure to the membrane surface at different stages of its membrane penetration. Aim 2 will be accomplished by characterizing the ability of native toxin to induce the insertion of the transmembrane B-hairpins (TMHs) of PFO mutants that alone can form an oligomeric prepore, but cannot insert their TMHs. In aim 3 the monomer-monomer interfaces of FF0 in the membrane-bound oligomeric complex will be revealed by the lack of accessibility to aqueous and membrane-restricted collisional quenchers of a fluorescent probe that will be placed at various locations on the surface of the monomer in cysteine-substituted derivatives of PFO. The location of the residue at an interface will be confirmed by site-specific crosslinking. Finally, we will investigate the intriguing property of intermedilysin, a member of the CDC family, which restricts its erythrocyte specificity to human erythrocytes, in contrast to all other known CDCs. We will identify and characterize the receptor for intermedilysin by a combination of receptor blots and affinity purification methods.